EP4054599A1 - Allogeneic t-cells and methods for production thereof - Google Patents
Allogeneic t-cells and methods for production thereofInfo
- Publication number
- EP4054599A1 EP4054599A1 EP20883680.9A EP20883680A EP4054599A1 EP 4054599 A1 EP4054599 A1 EP 4054599A1 EP 20883680 A EP20883680 A EP 20883680A EP 4054599 A1 EP4054599 A1 EP 4054599A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- cell line
- promoter
- cells
- nuclease
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
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Definitions
- the present invention provides methods for producing allogeneic T-cells, including the use of an engineered nuclease under the control of a controllable promoter.
- an engineered nuclease under the control of a controllable promoter.
- large volumes of cells can be prepared, each of which contains the ability to individually produce the desired nuclease.
- These cells can then be modified as desired through the introduction of a gene of interest, or an undesired gene can be knocked-out.
- allogeneic T-cells for use in various therapeutic applications.
- CAR T-Cell therapies One of the challenges facing CAR T-Cell therapies is the generation of allogenic cells that can be used for any patient. Scale up of allogeneic T-cell therapies can be prohibitively expensive and require long lead times due to the need for high quantities of viral vectors and/or recombinant endonucleases.
- a method of producing a T-cell line for use in an allogeneic application comprising: introducing a nucleic acid molecule encoding an engineered nuclease under the control of a controllable promoter into a T-cell line; integrating the nucleic acid molecule into the genome of the T-cell line; and expanding the T-cell line.
- a method of producing a genetically modified T- cell line comprising: introducing a nucleic acid molecule encoding a CRISPR-associated nuclease under the control of a controllable promoter into a T-cell line; integrating the nucleic acid molecule into the genome of the T-cell line; expanding the T-cell line; inducing expression of the CRISPR- associated nuclease by activating the controllable promoter; introducing a guide-RNA and a gene of interest into the expanded T-cell line; knocking out expression of a T-cell receptor and introducing the gene of interest into the genome of the T-cell line; and recovering the genetically modified T-cell line.
- a method of producing a chimeric antigen receptor (CAR) T- cell line comprising: introducing a nucleic acid molecule encoding a Cas9 nuclease under the control of a controllable promoter into a T-cell line; integrating the nucleic acid molecule into the genome of the T-cell line; expanding the T-cell line; inducing expression of the Cas9 nuclease by activating the controllable promoter; introducing a guide-RNA and a nucleic acid encoding a chimeric antigen receptor (CAR) into the expanded T-cell line; knocking out expression of a T- cell receptor and introducing the nucleic acid encoding the CAR into the genome of the T-cell line; and recovering the CAR T-cell line.
- CAR chimeric antigen receptor
- an allogeneic T-cell line comprising a CRISPR-associated (Cas) nuclease under the control of a controllable promoter integrated into the genome of the T-cell line.
- Cas CRISPR-associated
- FIG. 1 shows a schematic representation of both autologous and allogeneic approaches to T-cell therapies.
- FIG. 2 shows steps in the production of allogeneic T-cells.
- FIG. 3 shows three exemplary phases of allogeneic T-cell production.
- FIGS. 4A-4B show an exemplary derepressible promoter system for use herein.
- FIGS. 4C-4D shows an inducible vector system for use in embodiments hereof.
- FIG. 4E shows the TRE3G Tet-On system for use in embodiments hereof.
- FIGS. 5A-C show three treatment protocols for transduction of T-cells with a Cas9 inducible vector.
- FIGS. 6A-6B show the results of transduction of T-cells with a Cas9 inducible vector. See symbols on legend to track line-graphs.
- FIGS. 7A-7B shows the number of viable cells after selection, and during cell expansion.
- FIG. 8 shows measurement of exhaustion makers, senescence markers, activation markers, and T-cell markers for the three treatment protocols.
- FIG. 9 shows the results of induction of Cas9 expression in T-cells.
- FIGS. 10A and 10B show T-cell expansion after cryopreservation.
- FIG. 11 shows three approaches for TRAC gene knockout.
- FIGS. 12A-12C show TRAC knock-out 4 days post nucleofection.
- FIGS. 13A-13C show TRAC knock-out 7 days post nucleofection.
- FIGS. 14A-14C show TRAC knock-out 14 days post nucleofection.
- FIGS. 15A-15B show a compilation of knock-out experiments. DESCRIPTION OF THE INVENTION
- the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open- ended and do not exclude additional, unrecited, elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, system, host T-Cells, expression vectors, and/or composition of the invention. Furthermore, compositions, systems, cells, and/or nucleic acids of the invention can be used to achieve any of the methods as described herein.
- a chimeric antigen receptor T-cell is a T-cell (also called T Cell herein) that is modified with a chimeric antigen receptor (CAR) to more specifically target cancer cells.
- a CAR includes three parts: the ectodomain, the transmembrane domain, and the endodomain.
- the ectodomain is the region of the receptor that is exposed to extracellular fluid and includes three parts: a signaling peptide, an antigen recognition region, and a spacer.
- the signaling peptide directs the nascent protein into the endoplasmic reticulum.
- the signaling peptide is a single-chain variable fragment (scFv).
- the scFv includes the variable fragments of the light chain, connected with a short linker peptide.
- the linker includes glycine and serine.
- the linker includes glutamate and lysine.
- the transmembrane domain of the CAR is a hydrophobic a-helix that spans the membrane.
- the transmembrane domain of a CAR is a CD28 transmembrane domain.
- the CD28 transmembrane domain results in a highly expressed CAR.
- the transmembrane domain of a CAR is a O ⁇ 3-z transmembrane domain.
- the C/D3 -z transmembrane domain results in a CAR that is incorporated into a native T-cell receptor.
- the endodomain of the CAR is generally considered the “functional” end of the receptor. After antigen recognition by the antigen recognition region of the ectodomain, the CARs cluster, and a signal is transmitted to the cell.
- the endodomain is a C/D3 -z endodomain, which includes 3 immunoreceptor tyrosine-based activation motifs (ITAMs).
- ITAMs transmit an activation signal to the T-cell after antigen binding, triggering a T- cell immune response.
- Additional CAR designs known in the art can also be utilized in the practice of the methods described herein.
- CAR T-cells are removed from a human subject, genetically altered, and re-introduced into a patient to attack the cancer cells.
- CAR T-Cells can be derived from either the patient’s own blood (autologous), or derived from another healthy donor (allogenic). In general, CAR T-cells are developed to be specific to an antigen that is overexpressed on a tumor relative to healthy cells.
- T-cell that includes within the cell an engineered nuclease under the control of a controllable promoter, but still retain the natural T-cell receptor allowing for expansion.
- a desired CAR can then be inserted, the T-cell receptor removed, and the cells take for further processing and suitably for injection into a patient.
- FIG. 1 shows a schematic representation of both autologous and allogeneic approaches to T-cell therapies.
- T-cells are isolated from the patient, the CAR construct is virally transduced into a patient’s T-cells, and the CAR T-cells are then introduced back into the same patient.
- an allogeneic approach T-cells are isolated from healthy donors, the cells are virally transduced with the desired CAR construct.
- the T-cell receptor is knocked out to prevent graft vs. host disease (GvHD), a prerequisite for universal CAR T therapy (additional genes can also be knocked-out, including B2M and PD1 to help prevent GvHD).
- GvHD graft vs. host disease
- the allogeneic T-cells that include the desired CAR, can now be introduced into any patient. As described herein, producing a T-cell source that can be expanded prior to introduction of the CAR construct would allow for significant increases in scale-up, and also reduce needed resources and costs.
- T-cell line refers to lymphocyte cells developed in the thymus gland and that include a T-cell receptor on their surface. T-cells include immortalized T-cells.
- allogeneic or allogenic application refers to the use of cells from one or more donor sources (often a healthy donor) in therapeutic applications to one or more patients, which can be unrelated to the donor source(s).
- the methods described herein include introducing a nucleic acid molecule encoding an engineered nuclease under the control of a controllable promoter into a T- cell line.
- Methods of introducing a nucleic acid molecule into a T-cell line include the use of various transduction or transfection systems, including various viral systems, for example, the nucleic acid molecule can be introduced into the T-cell line using a lentiviral vector. Additional transduction or transfection systems include nucleofection, use of exosome systems, use of liposome systems, use of polymeric-based systems, etc.
- nucleic acid means a polymeric compound comprising covalently linked nucleotides.
- the term “nucleic acid” includes polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), both of which may be single- or double-stranded.
- DNA includes, but is not limited to, complimentary DNA (cDNA), genomic DNA, plasmid or vector DNA, and synthetic DNA.
- RNA includes, but is not limited to, mRNA, tRNA, rRNA, snRNA, microRNA, miRNA, or MIRNA.
- Nucleic acid also includes RNA that is introduced into a cell, and then is reverse transcribed to DNA, prior to being integrated into the genome of a call.
- a “gene” as used herein refers to an assembly of nucleotides that encode a polypeptide, and includes cDNA and genomic DNA nucleic acid molecules. “Gene” also refers to a nucleic acid fragment that can act as a regulatory sequence preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. In some embodiments, genes are integrated with multiple copies. In some embodiments, genes are integrated at predefined copy numbers.
- Transfection means the introduction of an exogenous nucleic acid molecule, including a vector, into a cell.
- a “transfected” cell comprises an exogenous nucleic acid molecule inside the cell and a “transformed” cell is one in which the exogenous nucleic acid molecule within the cell induces a phenotypic change in the cell.
- the transfected nucleic acid molecule can be introduced into the cell as RNA, reverse transcribed by the cell into DNA, and then integrated into the host T-Cell's genomic DNA and/or can be maintained by the cell, temporarily or for a prolonged period of time, extra-chromosomally.
- Host T-Cells or organisms that express exogenous nucleic acid molecules or fragments are referred to as “recombinant,” “transformed,” or “transgenic” organisms.
- transfection techniques are generally known in the art. See, e.g., Graham et ah, Virology, 52:456 (1973); Sambrook et ah, Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York (1989); Davis et ah, Basic Methods in Molecular Biology, Elsevier (1986); and Chu et al., Gene 73:197 (1981).
- transfection of a T-cell with one or more of the vectors described herein utilizes a transfection agent, such as polyethylenimine (PEI) or other suitable agent, including various lipids and polymers, to integrate the nucleic acids into the host T-CelFs genomic DNA.
- a transfection agent such as polyethylenimine (PEI) or other suitable agent, including various lipids and polymers, to integrate the nucleic acids into the host T-CelFs genomic DNA.
- the transfection includes viral infection (also termed “transduction”), transposons, mRNA transfection, electroporation, or combinations thereof.
- the transfection includes electroporation.
- the transfection includes viral transduction.
- the vector may be a viral vector, such as, for example, a lentiviral vector, a gammaretroviral vector, an adeno-associated viral vector, or an adenoviral vector.
- the transfection includes introducing a viral vector into the activated T cells of the cell culture.
- the vector is delivered as a viral particle.
- T-cells are suitably contacted with a nucleic acid molecule contained within a viral particle to allow for the nucleic acid to integrate into the genome of the T- cell line.
- the nucleic acid is introduced as RNA, is reverse transcribed by the cell to DNA, and is then integrated into the genome of the cell.
- This provides a T-cell line that includes within each cell, a genomically integrated engineered nuclease, under the control of a controllable promoter.
- These T-cells can then be expanded as described herein to produce the T-cell line for use in an allogeneic application.
- nuclease refers to a nuclease that has been separated, modified, mutated, and/or altered from it’s natural state as a nuclease.
- a “nuclease” refers to an enzyme that is able to cut a DNA and/or RNA molecule. By engineering the nuclease, the specific location of the cut can be designed and tailored to the desired cell type and/or gene of interest.
- Exemplary engineered nucleases that can be inserted into the T-cells include, for example, a meganuclease, a methyltransferase a zinc finger nuclease, a transcription activator-like effector-based nuclease (TALENS), a Fokl nuclease, and a CRISPR-associated nuclease.
- engineered nucleases use a DNA-binding protein which has both a desired catalytic activity and the ability to bind the desired target sequence through a protein-nucleic-acid interaction in a manner similar to restriction enzymes.
- Examples include meganucleases which are naturally occurring or engineered rare sequence cutting enzymes, zinc finger nucleases (ZFNs) or transcription activator-like nucleases (TALENs) which contain the Fokl catalytic nuclease subunit linked to a modified DNA binding domain and can cut one predetermined sequence each.
- ZFNs zinc finger nucleases
- TALENs transcription activator-like nucleases
- the binding domain is comprised of chains of amino-acids folding into customized zinc finger domains.
- TALENs similarly, 34 amino acid repeats originating from transcription factors fold into a huge DNA-binding domain.
- these enzymes can cleave genomic DNA to form a double strand break (DSB) or create a nick which can be repaired by one of two repair pathways, non-homologous end joining (NHEJ) or homologous recombination (HR).
- the NHEJ pathway can potentially result in specific mutations, deletions, insertions or replacement events.
- the HR pathway results in replacement of the targeted sequence by a supplied donor sequence.
- Exemplary Fokl and methyltransferase-based systems are described in U.S. Patent No. 10,220,052, the disclosure of which is incorporated by reference herein in its entirety.
- the CRISPR-associated nuclease is a Cas9 nuclease, or can be other Cas nucleases such as Casl2 nuclease, Casl3 nuclease, Casl4 nuclease, etc.
- the Cas9 nuclease is a Cas9 nuclease that has reduced immunogenicity, such as disclosed in U.S. Published Patent Application No. 2018-0319850, the disclosure of which is incorporated by reference herein in its entirety.
- CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
- Cas proteins CRISPR-associated nucleases, or Cas proteins
- the main defining features of the separate Types are the various cas genes, and the respective proteins they encode, that are employed.
- the casl and cas2 genes appear to be universal across the three main Types, whereas cas3, cas9, and cas 10 are thought to be specific to the Type I, Type II, and Type III systems, respectively. See, e.g., Barrangou, R. and Marraffini, L.A., "CRISPR-Cas systems: prokaryotes upgrade to adaptive immunity," Mol. Cell. 54(2):234-44 (2014), which is incorporated by reference herein in its entirety.
- the CRISPR-Cas system functions by capturing short regions of invading viral or plasmid DNA and integrating the captured DNA into the host genome to form so-called CRISPR arrays that are interspaced by repeated sequences within the CRISPR locus. This acquisition of DNA into CRISPR arrays is followed by transcription and RNA processing.
- CRISPR RNA processing proceeds differently.
- the transcribed RNA is paired with a transactivating RNA (tracrRNA) before being cleaved by RNase III to form an individual CRISPR-RNA (crRNA).
- the crRNA is further processed after binding by the Cas9 nuclease to produce the mature crRNA.
- the crRNA/Cas9 complex subsequently binds to DNA containing sequences complimentary to the captured regions (termed protospacers).
- the Cas9 protein then cleaves both strands of DNA in a site-specific manner, forming a double-strand break (DSB).
- RNA small guide RNA
- the CRISPR-Cas9 gene editing system has been used successfully in a wide range of organisms and cell lines, both in order to induce double-strand break formation using the wild type Cas9 protein or to nick a single DNA strand using a mutant protein termed Cas9n/Cas9 D10A (see, e.g., Mali et ah, (2013) and Sander and Joung, "CRISPR-Cas systems for editing, regulating and targeting genomes," Nature Biotechnology 32(4):347-55 (2014), each of which is incorporated by reference herein in its entirety).
- the Cas9n/Cas9 D10A nickase avoids indel creation (the result of repair through non-homologous end-joining) while stimulating the endogenous homologous recombination machinery.
- the Cas9n/Cas9 D10A nickase can be used to insert regions of DNA into the genome with high-fidelity.
- the CRISPR-associated nuclease that is inserted into the genome of the T-cell is a Cas9 nuclease.
- a Cas9 nuclease By placing the Cas9 nuclease under the control of a controllable promoter, the nuclease can be kept dormant or silent prior to its desired use as a gene editing tool.
- a “controllable promoter” refers to promoter that can be turned on or off, depending on the desired control of the gene that is under control of the promoter.
- Casl2, Casl3 and Casl4 nucleases can also be utilized in the methods described herein.
- Cast 2 nuclease creates staggered cuts in dsDNA (5 nucleotide 5' overhand dsDNA break).
- Casl2 processes its own guide RNAs, leading to increased multiplexing ability.
- Casl3t targets RNA, not DNA. Once it is activated by a ssRNA sequence bearing complementarity to its crRNA spacer, it unleashes a nonspecific RNase activity and destroys all nearby RNA regardless of their sequence.
- CRISPR-Casl2 and Casl3 the lesser known siblings of CRISPR Cas9
- Cell Biology and Toxicology pages 1-4 August 29, 2019
- an inactivated Cas9 enzyme can be linked to an active endonuclease and utilized in the methods described herein, including for example, a dCas9- Fokl fusion.
- under control refers to a gene being regulated by a “promoter,” “promoter sequence,” or “promoter region,” which refers to a DNA regulatory region/sequence capable of binding RNA polymerase and initiating transcription of a downstream coding or non coding gene sequence.
- the promoter and the gene are in operable combination or operably linked.
- the terms “in operable combination”, “in operable order” and “operably linked” refer to the linkage of nucleic acid sequences in such a manner that a promoter capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced.
- the term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced.
- the promoter sequence includes the transcription initiation site and extends upstream to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
- the promoter sequence includes a transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain “TATA” boxes and “CAT” boxes.
- promoters including inducible promoters, may be used to drive the gene expression, e.g., in the host T-Cell or vectors of the present disclosure.
- the promoter is not a leaky promoter, i.e., the promoter is not constitutively expressing any of the gene products as described herein.
- the promoter is a constitutive promoter, which initiates mRNA synthesis independent of the influence of an external regulation.
- the promoters used to control the engineered nucleases are inducible promoters.
- “Inducible promoters” refers to a group of promoters that can enhance the expression of exogenous genes under the stimulation of specific physical, chemical, or pathogen signals.
- Exemplary inducible promoters that can be used to control the engineered nuclease in embodiments hereof include, but are not limited to, a 4HT inducible promoter, a rapamycin inducible promoter, a hormone response element, a TET-on system, or a glutamate inducible promoter.
- the promoters used to control the engineered nucleases are derepressible promoters.
- a “derepressible promoter” refers to a structure that includes a functional promoter and additional elements or sequences capable of binding to a repressor element to cause repression of the functional promoter. “Repression” refers to the decrease or inhibition of the initiation of transcription of a downstream coding or non-coding gene sequence by a promoter.
- a “repressor element” refers to a protein or polypeptide that is capable of binding to a promoter (or near a promoter) so as to decrease or inhibit the activity of the promoter.
- a repressor element can interact with a substrate or binding partner of the repressor element, such that the repressor element undergoes a conformation change. This conformation change in the repressor element takes away the ability of the repressor element to decrease or inhibit the promoter, resulting in the “derepression” of the promoter, thereby allowing the promoter to proceed with the initiation of transcription.
- a “functional promoter” refers to a promoter, that absent the action of the repressor element, would be capable of initiation transcription.
- promoters that can be used in the practice of the present invention are known in the art, and include for example, PCMV, PHI, PI 9, P5, P40 and promoters of Adenovirus helper genes (e.g., El A, E1B, E2A, E40rf6, and VA).
- Exemplary repressor elements and their corresponding binding partners that can be used as derepressible promoters are known in the art, and include systems such as the cumate gene- switch system (CuO operator, CymR repressor and cumate binding partner) (see, e.g., Mullick et ah, “The cumate gene-switch: a system for regulated expression in mammalian cells,” BMC Biotechnology 6:43 (1-18) (2006), the disclosure of which is incorporated by reference herein in its entirety, including the disclosure of the derepressible promoter system described therein) and the TetO/TetR system described herein (see, e.g., Yao et ah, “Tetracycline Repressor, tetR, rather than the tetR-Mammalian Cell Transcription Factor Fusion Derivatives, Regulates Inducible Gene Expression in Mammalian Cells,” Human Gene Therapy 9:1939-1950 (1998), the disclosure of
- the derepressible promoters comprise a functional promoter and either one two tetracycline operator sequences (TetO or TetCk).
- the nucleic acid introduced into the T-cells further includes a tetracycline repressor protein to control the TetO derepressible system.
- T-cells in phase 1, can be transfected with an inducible Cas9 (iCas9) (or Cas9 under the control of a derepressible promoter) via a viral system, such as a lentivirus.
- iCas9 inducible Cas9
- a viral system such as a lentivirus
- the iCas9 T-cells (or T-cells containing another engineered nuclease), can be expanded using various methods for cell expansion.
- Expansion methods of the T-cells described herein can utilize any suitable reactor(s) including but not limited to stirred tank bioreactor, airlift, fiber, microfiber, hollow fiber, ceramic matrix, fluidized bed, fixed bed, and/or spouted bed bioreactors.
- reactor can include a fermenter or fermentation unit, or any other reaction vessel and the term “reactor” is used interchangeably with “fermenter.”
- fermenter or fermentation refers to both microbial and mammalian cultures.
- an example bioreactor unit can perform one or more, or all, of the following: feeding of nutrients and/or carbon sources, injection of suitable gas (e.g., oxygen), inlet and outlet flow of fermentation or cell culture medium, separation of gas and liquid phases, maintenance of temperature, maintenance of oxygen and CO2 levels, maintenance of pH level, agitation (e.g., stirring), and/or cleaning/sterilizing.
- suitable gas e.g., oxygen
- Example reactor units such as a fermentation unit, may contain multiple reactors within the unit, for example the unit can have 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100, or more bioreactors in each unit and/or a facility may contain multiple units having a single or multiple reactors within the facility.
- the bioreactor can be suitable for batch, semi fed-batch, fed-batch, perfusion, and/or a continuous fermentation processes. Any suitable reactor diameter can be used. In embodiments, the bioreactor can have a volume between about 100 mL and about 50,000 L.
- Non-limiting examples include a volume of 100 mL, 250 mL, 500 mL, 750 mL, 1 liter, 2 liters, 3 liters, 4 liters, 5 liters, 6 liters, 7 liters, 8 liters, 9 liters, 10 liters, 15 liters, 20 liters, 25 liters, 30 liters, 40 liters, 50 liters, 60 liters, 70 liters, 80 liters, 90 liters, 100 liters, 150 liters, 200 liters, 250 liters, 300 liters, 350 liters, 400 liters, 450 liters, 500 liters, 550 liters, 600 liters, 650 liters, 700 liters, 750 liters, 800 liters, 850 liters, 900 liters, 950 liters, 1000 liters, 1500 liters, 2000 liters, 2500 liters, 3000 liters, 3
- suitable reactors can be multi-use, single-use, disposable, or non-disposable and can be formed of any suitable material including metal alloys such as stainless steel (e.g., 316L or any other suitable stainless steel) and Inconel, plastics, and/or glass.
- the expansion suitably includes activation of the T-cells.
- metal alloys such as stainless steel (e.g., 316L or any other suitable stainless steel) and Inconel, plastics, and/or glass.
- the expansion suitably includes activation of the T-cells.
- metal alloys such as stainless steel (e.g., 316L or any other suitable stainless steel) and Inconel, plastics, and/or glass.
- the expansion suitably includes activation of the T-cells.
- APCs antigen-presenting cells
- TCR T-Cell Receptor
- MHC major histocompatibility complex
- TCR associates with CD3, a T-Cell co-receptor that helps to activate both cytotoxic T-Cells (e.g., CD8+ naive T-Cells) and T helper cells (e.g., CD4+ naive T-Cells).
- cytotoxic T-Cells e.g., CD8+ naive T-Cells
- T helper cells e.g., CD4+ naive T-Cells.
- T-Cell activation follows a two-signal model, requiring stimulation of the TCR/CD3 complex as well as a co-stimulatory receptor.
- Non-limiting examples of co-stimulatory molecules for T-Cells include CD28, which is a receptor for CD80 and CD86 on the membrane of APC; and CD278 or ICOS (Inducible T-cell COStimulator), which is a CD28 superfamily molecule expressed on activated T-Cells that interacts with ICOS-L.
- the co-stimulatory molecule is CD28.
- the co-stimulatory molecule is ICOS.
- the co-stimulatory signal can be provided by the B7 molecules on the APC, which bind to the CD28 receptor on T-Cells.
- B7 is a peripheral transmembrane protein found on activated APCs that can interact with CD28 or CD 152 surface proteins on a T-Cell to produce a co-stimulatory signal.
- the co-stimulatory molecule is B7.
- a T-Cell culture is activated with an activation reagent.
- the activation reagent is an Antigen-Presenting Cell (APC).
- the activation reagent is a dendritic cell. Dendritic cells are APCs that process antigen and present it on the cell surface to T-Cells.
- the activation reagent is co-cultured with the T-Cell culture. Co-culturing may require separate purification and culturing of a second cell type, which may increase labor requirements and sources of variability. Thus, in some embodiments, alternative activation methods are used.
- the activation reagent is an antibody.
- the cell culture is activated with an antibody bound to a surface, including a polymer surface, including a bead.
- the one or more antibodies is an anti-CD3 and/or anti-CD28 antibody.
- the beads may be magnetic beads such as, e.g., DYNABEADS, coated with anti-CD3 and anti-CD28.
- the anti-CD3 and anti-CD28 beads can suitably provide the stimulatory signals to support T-Cell activation. See, e.g., Riddell 1990; Trickett 2003.
- the cell culture is activated with a soluble antibody.
- the soluble antibody is a soluble anti-CD3 antibody.
- OKT3 is a murine monoclonal antibody of the immunoglobulin IgG2a isotype and targets CD3.
- the soluble anti-CD3 antibody is OKT3.
- OKT3 is further described in, e.g., Dudley 2003; Manger 1985; Ceuppens 1985; Van Wauwe 1980; Norman 1995.
- the co-stimulatory signal for T-Cell activation is provided by accessory cells.
- Accessory cells may include, for example, a Fc receptor, which enables cross- linking of the CD3 antibody with the TCR/CD3 complex on the T-Cell.
- the cell culture is a mixed population of peripheral blood mononuclear cells (PBMCs).
- PBMC peripheral blood mononuclear cells
- PBMC may include accessory cells capable of supporting T-Cell activation.
- CD28 co-stimulatory signals can be provided by the B7 molecules present on monocytes in the PBMC.
- the accessory cells include a monocyte or a monocyte-derived cell (e.g., a dendritic cell).
- the accessory cells include B7, CD28, and/or ICOS. Accessory cells are further described in, e.g., Wolf 1994; Chai 1997; Verwilghen 1991; Schwartz 1990; Ju 2003; Baroja 1989; Austyn 1987; Tax 1983.
- activation reagent may determine the phenotype of the CAR T- Cells produced, allowing for the promotion of a desired phenotype.
- the activation reagent determines the ratio of T-Cell subsets, i.e., CD4+ helper T-Cells and CD8+ cytotoxic T-Cells.
- the cytotoxic CD8+ T-Cells are typically responsible for killing cancer cells (i.e., the anti-tumor response), cells that are infected (e.g., with viruses), or cells that are damaged in other ways.
- CD4+ T-Cells typically produce cytokines and help to modulate the immune response, and in some cases may support T-Cell lysis.
- the methods of the present disclosure further include producing CAR T-Cells of a pre-defmed phenotype (i.e., promoting cells of a desired phenotype).
- the pre-defmed phenotype may be, for example, a pre-defmed ratio of CD8+ cells to CD4+ cells.
- the ratio of CD8+ cells to CD4+ cells in a population of CAR T-Cells is about 1:1, about 0.25:1, or about 0.5:1.
- the ratio of CD8+ cells to CD4+ cells in a population of CAR T-Cells is about 2:1, about 3:1, about 4:1, or about 5:1.
- the T cell culture is expanded to a pre-defmed culture size (i.e., number of cells).
- the pre-defmed culture size may include a sufficient number of cells suitable for clinical use, i.e., transfusion into a patient, research and development work, etc.
- a clinical or therapeutic dose of T-cells for administration to a patient is about 10 5 cells, about 10 6 cells, about 10 7 cells, about 10 8 cells, about 10 9 cells, or about 10 10 cells.
- the method produces at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 clinical doses of T-cells (and thus ultimately CAR T-cells).
- the number of T-cells produced by the methods described herein is at least about 100 million (i.e., 100* 10 6 ) cells, or at least about 1 billion (i.e., 1*10 9 ) cells, at least about 50 billion, at least about 100 billion, at least about 250 billion, at least about 500 billion, at least about 750 billion, or at least about 1 trillion (i.e., 1*10 12 ) cells, including at least about 2 trillion, at least about 3 trillion, at least about 4 trillion, at least about 5 trillion, or at least about 10 trillion T-cells.
- the cells are prepared for storage, including for example, freezing the expanded T-cell line following the expanding.
- Methods of freezing the expanded cells are known in the art, and include the use of liquid nitrogen, dry ice, and can include various lyophilization procedures.
- the cells are frozen at a temperature of about -80°C to about 0°C, and can include the use of a cryoprotective agent such as dimethylsulfoxide (DMSO).
- DMSO dimethylsulfoxide
- a method of producing a genetically modified T-cell line comprising: introducing a nucleic acid molecule encoding a CRISPR- associated nuclease under the control of a controllable promoter into a T-cell line; integrating the nucleic acid molecule into the genome of the T-cell line; and expanding the T-cell line.
- the nucleic acid molecule encoding a CRISPR-associated nuclease is RNA that is reverse transcribed to DNA, and then integrated into the genome of the cell.
- the CRISPR-associated nuclease is a Cas9 nuclease or a Casl2 nuclease.
- the T-cell can be frozen if desired and stored.
- the stored cells are then suitably thawed prior to further processing.
- the methods can further include inducing expression of the CRISPR-associated nuclease by activating the controllable promoter.
- an inducible promoter such as a 4HT inducible promoter, a rapamycin inducible promoter, a hormone response element, or a glutamate inducible promoter
- the promoter is induced by the addition of, for example, 4-hydroxytamoxifen, rapamycin, a hormone, or glutamate, respectively.
- a derepressible promoter such as the TetO sequence described herein coupled to a CMV promoter
- the addition of doxycycline removes the repression, and allows the gene (engineered nuclease) to be expressed via the CMV promoter.
- the nucleic acid molecule that encodes the Cas9 also encodes a TetR repressor element, suitably under the control of another promoter system, such as a constitutive promoter like the hPGK promoter.
- the controllable promoter can also be a Tet on system, including the use of a TRE3G promoter sequence, as described herein.
- FIGs. 4A-4B illustrates an exemplary derepressible system, the TetO system described herein.
- two TetO sequences (along with a promoter sequence) are suitably oriented prior to the engineered nuclease (EN).
- EN engineered nuclease
- TetR tetracycline repressor protein
- the promoters e.g., CMV
- TetR suitable Doxycycline (Dox)
- the TetR proteins change conformation, release from the TetO sequences, and the functional promoters (e.g., CMV) begin its normal transcription process, as they would naturally, resulting in production of the engineered nuclease (EN).
- Dox Doxycycline
- FIG. 4C shows an exemplary inducible vector (e.g., a lentiviral vector) that can be used to integrate the inducible engineered nuclease, in this case a Cas9 nuclease under the control of a TET-on operating system (TRE3G), allowing for expression of the Cas9 in the cell upon induction with Doxycycline.
- FIG. 4D shows a more detailed vector map.
- FIG. 4E shows the operation of the TET-on operating systems, TRE3G. As shown, the Tet-On 3G transactivator protein, in the absence of doxy cy cline, does not bind to the TRE3G promoter sequence.
- the Tet-On 3G transactivator protein is able to bind to the TRE3G promoter sequence, which activates transcription, and causes the expression of Cas9 nuclease (or other nuclease if desired).
- controllable systems described herein for introducing a nuclease also suitably include a selectable marker, such as an antibiotic resistance gene (e.g., Ampicillin resistance) to allow for production of inducible-nuclease containing cells (including T-cells) that can readily be selected and enriched.
- a selectable marker such as an antibiotic resistance gene (e.g., Ampicillin resistance) to allow for production of inducible-nuclease containing cells (including T-cells) that can readily be selected and enriched.
- the other controllable systems described herein can also be used in combination with selectable markers to allow for selection of nuclease expressing cells, and then enrichment to provide a pure cell population with the desired nuclease (e.g., Cas9 or Casl2) integrated into the genome.
- phase 3 in addition to activating the expression of the Cas9 nuclease, a guide-RNA and a gene of interest are also introduced into the expanded T-cell line. As described herein, this introduction of the guide-RNA and gene of interest can be introduced via a transfection mechanism such as nucleofection.
- the T-cell receptors are suitably knocked out, and the gene of interest is introduced into the genome of the T-cell line.
- the T-cells are suitably expanded, then the genetically modified T-cell line is recovered.
- Methods for expansion are known in the art and described here. Methods of recovering the desired cells include various filtration methods, centrifugation, as well as cell isolation and washing.
- Knocking out the T-cell receptors suitably includes knocking out the TRAC gene (T- cell receptor alpha subunit), which leads to the ablation of the entire T-cell receptor.
- TRAC gene T- cell receptor alpha subunit
- various guide-RNA sequences can be used to knock out the TRAC gene, and includes those noted in the Examples, as well as other sequences that are readily determined by those of ordinary skill in the art.
- the gene of interest suitably encodes a chimeric antigen receptor (CAR). As shown in FIG.
- the genetic editing by the Cas9 nuclease results in the knocking out of the T-Cell receptor, and the expression of the desired CAR on the T-cell.
- Such T-cells can now be administered to the desired patient population, based on the desired CAR.
- Integration of the desired CAR construct into the T-cells suitably occurs at the location of the knock-out of the TRAC gene, such that the CAR is under the control of the endogenous promoter of the TRAC gene.
- the ability to expand the T-cells to significant numbers prior to inducing the expression of the Cas9, and then subsequent integration of the gene of interest allows for production of high number of T-cells, on the order of 10 9 -10 12 T cells.
- genes can be knocked out in the T-cells to create the desired modifications.
- genes such as programmed cell death protein 1 (PD1) and/or B2M gene (b2 microglobulin) responsible for encoding a serum protein found in association with the major histocompatibility complex (MHC) class I heavy chain. Knock-out of such genes can occur using various methods such as antisense, siRNA, microRNA, and other approaches known in the art.
- a method of producing a chimeric antigen receptor (CAR) T-cell line comprising: introducing a nucleic acid molecule encoding a Cas9 nuclease under the control of a controllable promoter into a T-cell line; integrating the nucleic acid molecule into the genome of the T-cell line; expanding the T-cell line; inducing expression of the Cas9 nuclease by activating the controllable promoter; introducing a guide-RNA and a nucleic acid encoding a chimeric antigen receptor (CAR) into the expanded T-cell line; knocking out expression of a T-cell receptor and introducing the nucleic acid encoding the CAR into the genome of the T-cell line; and recovering the CAR T-cell line.
- CAR chimeric antigen receptor
- controllable promoters including inducible promoters and derepressible promotors are described herein, as are methods of inducing expression of the Cas9 nuclease via the introduction of a molecule that induces expression, or that derepresses a derepressible promoter.
- an allogeneic T-cell line comprising a CRISPR-associated (Cas) nuclease under the control of a controllable promoter integrated into the genome of the T-cell line.
- the Cas nuclease is suitably a Cas9 nuclease.
- the allogenic T-cell line includes a controllable promoter that is an inducible promoter, including for example a 4HT inducible promoter, a rapamycin inducible promoter, a hormone response element, or a glutamate inducible promoter.
- the controllable promoter can also be a Tet-on system.
- controllable promoter can be a derepressible promoter, such as the use of one or more tetracycline operator sequences (TetO).
- T-cell further includes a nucleic acid molecule encoding a tetracycline repressor protein.
- the allogenic T-cells prepared in accordance with the embodiments described allow for the production of at least about 10 9 T-cells, at least about 10 10 T-cells, at least about 10 11 T-cells, or in embodiments at least about 10 12 T-cells.
- Administration to a human subject can include, for example, inhalation, injection, or intravenous administration, as well as other administration methods known in the art.
- Treatment 1 included 24 hr activation with IL-2 at 15 ng/mL & CD3/CD28 on the day of T cells isolation.
- IL2+CD3/CD28 included 24 hr activation with IL-2 at 15 ng/mL & CD3/CD28 on the day of T cells isolation.
- Treatment 3 included 24 hr Activation with IL-2 at 15 ng/mL & CD3/CD28 on the day of T cells isolation. Following transduction, cells were treated with IL-2 at 15 ng/mL & CD3/CD28. Expansion was conducted in the presence of only IL-2 & IL-7.
- FIGS. 6A-6B Results of the transduction are provided in FIGS. 6A-6B.
- FIG. 6 A the percent of GFP positive cells using the treatment combination of CD3/CD28+IL-2 had the greatest transduction efficiency, with both the control vector (#) and vector that contained the Cas9 nuclease gene (@).
- Treatment with IL-2+IL-7 also showed a good transduction ($).
- FIG. 6B shows the dilution rate of the GFP positive population.
- FIG. 7A shows the number of viable cells for the three treatments described in this Example. As illustrated, treatment with IL- 2+IL-7 showed the most viable cells. Upon expansion for 12 days after selection, cells treated with CD3/CD28+IL-2 and IL-2+IL-7 both showed a high number of viable cells. (FIG. 7B).
- Cells were induced for 24 hours with 1 pg/mL of Doxy cy cline, and the protein lysate was analyzed by Western Blot. As shown in FIG. 9, cell growth with treatment 3 (IL-2+IL-7) showed higher expression of Cas9 upon induction, but cells treatment with CD3/CD28+IL-2 also showed expression of the Cas9 nuclease.
- FIG. 10A viability was nearly identical before and after cryopreservation for both treatments noted.
- FIG. 10B shows cell viability days after thawing, indicating that for both treatment, cells were able to proliferate successfully.
- FIG. 11 shows the three sgRNA sequences (SEQ ID NOS: 1-3) that were investigated, along with the regions they target in the translated TRAC gene (SEQ ID NO:4).
- sgRNA sequences were transfected into the T-cell using a nucleofection procedure. Briefly, lxlO 6 cells in 20 pL were transduced at room temperature with about 3.3 pg of sgRNA using a AMAXA P2 primary cell 4D nucleofector X kit and a EO- 115 program.
- Knock-out experiments were carried out with sgRNAs TRAC# 1 -3 (SEQ ID NOs: 1-3) and calibrated relative to CD-3.
- Cas9 T-cells (treated with CD3/CD28 & IL-2, IL-7 at 15 ng/mL, and then selected with Blasticidin at 15 pg/mL) were thawed on Day 1.
- Cas9 was induced with Doxycyclin (2 pg/mL for 24 hours).
- FACS analysis of CD-3 and TCRa expression levels were conducted.
- FIGS 12A and 12B show TRAC knock-out 4 days post nucleofection, with TRAC#1 - #3 sgRNA sequences.
- FIG. 12C shows a compilation of the results. As indicated, each of TRAC#1 -TRAC#3 resulted in about 41-47% knock out of the TRAC gene, with TRAC#2 sgRNA showing the highest amount of knock-out (47%).
- FIGS 13A and 13B show TRAC knock-out 7 days post nucleofection, with TRAC#1- #3 sgRNA sequences.
- FIG. 13C shows a compilation of the results. As indicated, each of TRAC#1 -TRAC#3 resulted in about 66-74% knock out of the TRAC gene, with TRAC#2 sgRNA showing the highest amount of knock-out (74%).
- FIGS 14A and 14B show TRAC knock-out 14 days post nucleofection, with TRAC#1- #3 sgRNA sequences.
- FIG. 14C shows a compilation of the results. As indicated, each of TRAC# 1 -TRAC#3 resulted in about 84-89% knock out of the TRAC gene, with TRAC#2 sgRNA showing the highest amount of knock-out (89%).
- FIGS. 15A-15B A complication of the 14 days of experiments are shown in FIGS. 15A-15B, illustrating the effective knock-out of the TRAC gene using the methods described herein.
- CAR knock-in experiments are to be conducted following the knock-out experiment described above with the following additions.
- gRNA towards the TRAC gene
- the addition of a DNA template is performed. This DNA template is designed to integrate into the TRAC locus at the sight of the double-strand break generated by the nuclease using a homologous recombination mechanism.
- Anti CD 19-CAR fused to a green fluorescent protein (GFP) molecule at the cytosolic side which can be easily detected using FACS.
- GFP green fluorescent protein
- Embodiment 1 is a method of producing a T-cell line for use in an allogeneic application, comprising: introducing a nucleic acid molecule encoding an engineered nuclease under the control of a controllable promoter into a T-cell line; integrating the nucleic acid molecule into the genome of the T-cell line; and expanding the T-cell line.
- Embodiment 2 includes the method of embodiment 1, wherein the engineered nuclease is selected from the group consisting of a meganuclease, a zinc finger nuclease, a transcription activator-like effector-based nuclease, and a CRISPR-associated nuclease.
- the engineered nuclease is selected from the group consisting of a meganuclease, a zinc finger nuclease, a transcription activator-like effector-based nuclease, and a CRISPR-associated nuclease.
- Embodiment 3 includes the method of embodiment 2, wherein the CRISPR-associated nuclease is a Cas9 nuclease or a Casl2 nuclease.
- Embodiment 4 includes the method of any one of embodiments 1-3, wherein the controllable promoter is an inducible promoter.
- Embodiment 5 includes the method of embodiment 4, wherein the inducible promoter is a 4HT inducible promoter, a rapamycin inducible promoter, a hormone response element, or a glutamate inducible promoter.
- the inducible promoter is a 4HT inducible promoter, a rapamycin inducible promoter, a hormone response element, or a glutamate inducible promoter.
- Embodiment 6 includes the method of any one of embodiments 1-3, wherein the controllable promoter is a derepressible promoter.
- Embodiment 7 includes the method of embodiment 6, wherein the derepressible promoter includes one or more tetracycline operator sequences (TetO).
- TetO tetracycline operator sequences
- Embodiment 8 includes the method of embodiment 7, wherein the nucleic acid molecule further includes a tetracycline repressor protein.
- Embodiment 9 includes the method of any one of claims 1-3, wherein the controllable promoter comprises a Tet-on system.
- Embodiment 10 includes the method of any one of embodiments 1-9, further comprising freezing the expanded T-cell line following the expanding.
- Embodiment 11 includes the method of any one of embodiments 1-10, wherein the nucleic acid molecule is introduced into the T-cell line using a lentiviral vector.
- Embodiment 12 includes the method of any one of embodiments 1-11, wherein the T- cell line comprises at least about 10 9 T-cells.
- Embodiment 13 is a method of producing a genetically modified T-cell line, comprising: introducing a nucleic acid molecule encoding a CRISPR-associated nuclease under the control of a controllable promoter into a T-cell line; integrating the nucleic acid molecule into the genome of the T-cell line; expanding the T-cell line; inducing expression of the CRISPR- associated nuclease by activating the controllable promoter; introducing a guide-RNA and a gene of interest into the expanded T-cell line; knocking out expression of a T-cell receptor and introducing the gene of interest into the genome of the T-cell line; and recovering the genetically modified T-cell line.
- Embodiment 14 includes the method of embodiment 13, wherein the CRISPR- associated nuclease is a Cas9 nuclease or a Casl2 nuclease.
- Embodiment 15 includes the method of embodiment 13 or embodiment 14, wherein the controllable promoter is an inducible promoter.
- Embodiment 16 includes the method of embodiment 15, wherein the inducible promoter is a 4HT inducible promoter or a glutamate inducible promoter.
- Embodiment 17 includes the method of embodiment 13 or embodiment 14, wherein the controllable promoter is a derepressible promoter.
- Embodiment 18 includes the method of embodiment 17, wherein the derepressible promoter includes one or more tetracycline operator sequences (TetO).
- TetO tetracycline operator sequences
- Embodiment 19 includes the method of embodiment 18, wherein the nucleic acid molecule further includes a tetracycline repressor protein.
- Embodiment 20 includes the method of embodiment 18 or embodiment 19, wherein the activating the derepressible promoter comprises adding doxycycline to the T-cell line.
- Embodiment 21 includes the method of embodiment 13 or 14, wherein the controllable promoter comprises a Tet-on system.
- Embodiment 22 includes the method of embodiment 21, wherein the activating the Tet on system comprises added doxycycline to the T-cell line.
- Embodiment 23 includes the method of any one of embodiments 13-22, wherein the gene of interest encodes a chimeric antigen receptor (CAR).
- CAR chimeric antigen receptor
- Embodiment 24 includes the method of any one of embodiments 13-23, further comprising freezing the T-cell line following the expanding in c, and thawing prior to the inducing in d.
- Embodiment 25 includes the method of any one of embodiments 13-24, wherein the genetically modified T-cell line comprises at least about 10 9 T-cells.
- Embodiment 26 is a method of producing a chimeric antigen receptor (CAR) T-cell line, comprising: introducing a nucleic acid molecule encoding a Cas9 nuclease under the control of a controllable promoter into a T-cell line; integrating the nucleic acid molecule into the genome of the T-cell line; expanding the T-cell line; inducing expression of the Cas9 nuclease by activating the controllable promoter; introducing a guide-RNA and a nucleic acid encoding a chimeric antigen receptor (CAR) into the expanded T-cell line; knocking out expression of a T-cell receptor and introducing the nucleic acid encoding the CAR into the genome of the T-cell line; and recovering the CAR T-cell line.
- CAR chimeric antigen receptor
- Embodiment 27 includes the method of embodiment 26, wherein the controllable promoter is an inducible promoter.
- Embodiment 28 includes the method of embodiment 27, wherein the inducible promoter is a 4HT inducible promoter or a glutamate inducible promoter.
- Embodiment 29 includes the method of embodiment 26, wherein the controllable promoter is a derepressible promoter.
- Embodiment 30 includes the method of embodiment 29 wherein the derepressible promoter includes one or more tetracycline operator sequences (TetO).
- Embodiment 31 includes the method of embodiment 30, wherein the nucleic acid molecule further includes a tetracycline repressor protein.
- Embodiment 32 includes the method of embodiment 30 or embodiment 31, wherein the activating the derepressible promoter comprises adding doxycycline to the T-cell line.
- Embodiment 33 includes the method of embodiment 26, wherein the controllable promoter comprises a Tet-on system.
- Embodiment 34 includes the method of embodiment 33, wherein the Tet-on system comprises adding doxycycline to the T-cell ine.
- Embodiment 35 includes the method of any one of embodiments 26-34, further comprising freezing the T-cell line following the expanding in c, and thawing prior to the inducing in d.
- Embodiment 36 includes the method of any one of embodiments 26-35, wherein the CAR T-cell line comprises at least about 10 9 T-cells.
- Embodiment 37 is an allogeneic T-cell line, comprising a CRISPR-associated (Cas) nuclease under the control of a controllable promoter integrated into the genome of the T-cell line.
- Cas CRISPR-associated
- Embodiment 38 includes the allogeneic T-cell line of embodiment 37, wherein the CRISPR-associated nuclease is a Cas9 nuclease or a Casl2 nuclease.
- Embodiment 39 includes the allogeneic T-cell line of embodiment 37 or embodiment 38, wherein the controllable promoter is an inducible promoter.
- Embodiment 40 includes the allogeneic T-cell line of embodiment 39, wherein the inducible promoter is a 4HT inducible promoter or a glutamate inducible promoter.
- Embodiment 41 includes the allogeneic T-cell line of embodiment 37 or embodiment 38, wherein the controllable promoter is a derepressible promoter.
- Embodiment 42 includes the allogeneic T-cell line of embodiment 41, wherein the derepressible promoter includes one or more tetracycline operator sequences (TetO).
- Embodiment 43 includes the allogeneic T-cell line of embodiment 42, wherein T-cell further includes a nucleic acid encoding a tetracycline repressor protein.
- Embodiment 44 includes the allogeneic T-cell line of embodiment 37 or embodiment 38, wherein the controllable promoter comprises a Tet-on system.
- Embodiment 45 includes the allogeneic T-cell line of any one of embodiments 37-44, comprising at least about 10 9 T-cells.
- Embodiment 46 includes the allogeneic T-cell line of embodiment 45, comprising at least about 10 10 T-cells.
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